我们衷心感谢茂雄的宝贵建议和讨论。我们还感谢约翰和 Sumire Eckstein 对手稿的批判性阅读。

注: 本研究中使用的标本为微生物, 在液氮中迅速冷冻, 用丙烷取代含有2% 锇氧化的丙酮, 并嵌入环氧树脂1,2,3, 4,5,6,7,8,9,10,11,12,13 ,14,15,16,17,18。
1. 支持膜的制备 (图 1-3)
注: 使用 cast-on-glass 方法19准备了银色的 Formvar 支持胶片。
<ol><li>在二氯化乙烯中加入100毫升 1.5% Formvar 的 Formvar 制造装置 (图 1)。通过用橡皮球19将溶液与空气通过 "a", 在该装置的上部柱上的 Formvar 溶液中浸 one-half (76 mm x 26 mm x 1.3 mm)。<ol>
<li>通过打开三路塞并通过 "b" 释放空气以减少压力, 从柱上排出溶液。取出装置上的玻璃片, 在空气中晾干, 在玻璃滑梯的表面形成薄膜。使用白炽灯加速干燥的 Formvar 膜。</li>
	</ol>
</li>
	<li>在用刀片 (图 2a) 将玻璃片上的四边缘刮掉后, 并在幻灯片上呼吸以方便胶片从幻灯片20中分离出来, 将玻璃片浸入水中, 使胶片漂浮在水缓慢地在低水平角度 (约10°,图 2b)。</li>
	<li>用铝架 (30 mm x 25 mm x 3 mm) 用孔 (直径 4 mm) 舀出 Formvar 膜 (图 3a)。将机架与 Formvar 胶片放在干燥中直到使用 (图 3b)。</li>
</ol>2. 在显微镜 (图 4) 下, 用超声波修剪刀片和刀片式服务器21修剪试样块
<ol><li>通过用光学显微镜 (图 4a) 观察块的尖端, 确保块表面有细胞。</li>
	<li>将试样块装入卡盘 (块座), 然后在修剪阶段安装卡盘21 (图 4b)。这种修剪阶段有一个从标本背面的照明机制。</li>
	<li>修剪块的大小为 0.7 mm x 1.0 mm (图 4c, 5d)。由于环氧树脂是非常硬, 修剪的方块首先使用超声波修剪刀片。超声波修边刀是一种新引进的机器, 其块体易于修剪。然后用剃刀刀片进一步修剪木块。剪切一个肩膀以标记切削方向 (请参见图 5d,图 8)。</li>
</ol>3. 使用切片用金刚石刀修整试样块 (图 5)
<ol><li>在 ultramicrotome 标本夹中设置试样块夹头。在执行连续超薄切片时, 将试样的90°逆时针放置到实际位置 (请参见图 5d)。</li>
	<li>用菱形修剪刀切割块的表面 (参见图 5d)。将菱形刀边与试样块面平行, 并切下试样表面的最小量, 以免丢失任何标本 (图 5d)。 注意: 这一步是做抚平的标本表面。细长部分的试样表面部分保持完好 (因此, 这部分不像镜子那样闪亮,图 5d), 这样就不会丢失样本的任何部分进行连续切片。这是因为标本是暴露在表面的块。</li>
	<li>在刀台上放置一面镜子 (台面切割, M), 以监视试样块的切割 (图 5c)。</li>
	<li>切割左边缘 (这成为上侧,图 5d, 在序列切片的样本) 的块使用修剪刀旋转刀阶段30°向左 (图 5a)。</li>
	<li>从试样的左边缘切下100µm 的块面 (这成为序列切片中试样的下部,图 5d), 通过将刀阶段30°向右旋转 (图 5b)。 注: 该系列的部分将削减约 90 nm x 约1毫米大小的标本薄部分。大的部分将用于调整试样表面和刀口, 使其平行。在将试样表面调整到与刀口平行的地方后, 将用刀片将大零件移除。通过使用切片切割试样的上下两侧, 试样的两侧变得平滑且完全平行 (图 5d), 这是获得平直和完整的带状剖面所必需的。</li>
</ol>4. 调整试样表面和刀口, 使其与其他21平行
<ol><li>取下裁切刀, 顺时针旋转试样块90°。</li>
	<li>把超薄切片刀放到刀台上</li>
	<li>调整试样表面和刀口, 使其与试样表面的大部分面平行。 注: 试样的大部分用于调整, 因为连续切片的切割面很小, 很难用这部分来调整试样表面和刀口, 特别是在垂直方向。因此, 使用的大部分标本, 使调整容易。</li>
</ol>5. 在试样块上散布氯丁橡胶溶液 (图 6)
<ol><li>要将标本夹头放置在与原始位置完全相同的位置, 请在卡盘和切片的卡盘上应用磁带, 并在边界处剪切 (图 6a)。</li>
	<li>从切片取出标本夹头, 并将其置于显微镜 (图 6b) 下。用剃刀刀片切断标本的大部分, 留下纤细的部分, 从中可以获得串行部分。</li>
	<li>使用巴斯德吸管, 下降约1µL 0.5% 氯丁橡胶解决方案 (图 6b) 上的标本, 以用于串行切片, 使节方胶粘剂。用氯丁橡胶溶液覆盖整个试样。立即吸收过剩的氯丁橡胶解决方案与一块滤纸放置在靠近标本。获得一条丝带的部分是必不可少的拍照的串行细胞部分, 和氯丁橡胶胶水是非常有用的, 以坚持节。</li>
	<li>准备3狭缝网格 (狭缝尺寸: 中间, 0.4 毫米 x 2.2 毫米, 两侧 0.2 mm x 2.2 mm) (图 6c) 通过将网格的手柄弯曲到60°, 用0.5% 氯丁橡胶溶液处理网格, 并使网格亲水性辉光放电22。</li>
</ol>6. 制作串行部分 (图 7-9)
<ol><li>将样本块卡盘放回切片, 并将其与 5.1 (图 6a) 对齐, 使其与录音部件对准。这使块面和刀边完全平行。然后把刀靠近标本。</li>
	<li>把刀船装满水。</li>
	<li>用塑料盖盖住切片以防止气流 (图 7)。 注: 在超薄切片和剖面提取过程中的气流通常会引起问题。塑料盖有三孔: 一个孔是为双目透镜, 另外两个孔是为胳膊允许操作, 当盖子是。木扶手是用来放置武器, 而做微妙的工作, 如检索串行部分与网格。木扶手被放置在不同的表从切片表 (图 7, 箭头), 以便不传送操作员的手的振动到切片。</li>
	<li>开始切割 200 nm 截面厚度的试样 (图 8)。设置在 200 nm 厚度将节省时间把刀的表面上的标本。</li>
	<li>剪切第一个截面后, 将截面厚度设置为 70 nm (图 8)。</li>
	<li>当连续区段的数量到达 20 (确切地说, 在丝带以后到达大约1.8 毫米; 区段的数量根据区段的宽度变化), 设置部分厚度到10毫微米 (图 8), 当继续切开。 注: 由于切片不能切割10纳米厚的截面, 没有新的节出现, 和以前的切割部分成为分离的刀口。重要的是要获得分离的节组 (图 9) 来拾取串行部分, 以避免使用一对头发线手动分离分区的困难。因为视野在传输电子显微镜是2.0 毫米, 部分应该是1.8 毫米长至多。</li>
	<li>将截面厚度设置为 60 nm (图 8)。这将生成 70 nm 剖面 (图 8), 因为该计算机将添加以前的 10 nm 厚度。</li>
	<li>将截面厚度设置回 70 nm (图 8), 并继续切削, 直到获得 1.8 mm 长的剖面。</li>
	<li>重复 6.6-6.8, 直到五 1.8 mm 长剖面得到 (图 9)。我们通常做五节组, 因为有五标本持有人在透射电子显微镜可在我们的实验室, 但五不是极限。</li>
</ol>7. 拾取串行部分 (图 10-12)
<ol><li>将 "节保持环路"23 (循环的内径: 5.0 mm) (图 10a) 放在第三个分区组 (图 10b) 上。 注意: 有必要按其切片顺序检索各节以按正确顺序拍摄图片。然而, 由于在刀船上有五节组, 它往往会混淆哪些群体是哪一组。通过将循环放在第三个组上, 就可以清楚地看出, 运算符附近的两个组是第一和第二类, 而从运算符到距离的两个群是第四和第五组 (图 10b)。这将防止混合订单。</li>
	<li>将 "水面提升回路"23 (WSRL, 循环的内径: 4.0 mm) (图 11a) 放在刀台上 (图 11b)。WSRL 将坚定地站在刀台上, 因为它在底部装有磁铁。<ol>
<li>通过移动轴和手柄, 将 WSRL 的环路放在串行节的上方。把 WSRL 的环放在水面上, 这样循环就会包围这些部分。</li>
		<li>通过将螺钉向下转动来推动循环, 使水面表面张力升高 (图 11 c-d)。将剖面移动到循环的中心, 将其放置在水的顶点上, 并使用发束21调整剖面的方向。</li>
		<li>拿起部分组通过触摸它与一个裸露的狭缝网格, 这是由镊子举行, 并保持平行的水面 (图 11d)。对于部分来说, 首先接触网格 (而不是水), 将剖面精确地放置在网格的中心 (图 11d) 是很重要的。</li>
	</ol>
</li>
	<li>将网格与剖面一起放置在 Formvar 支持膜上的小水滴16 (图 12), 并使用滤纸去除多余的水。 注: 由于膜 (切片) 直接触及膜 (支撑膜), 支撑膜的皱起通常是一个问题。通过在 Formvar 支持膜上放置一个带截面的网格和少量的水来减少支撑膜上的皱纹的问题,16。</li>
</ol>8. 着色部分16,24 (图 13)
<ol><li>在切片完全干燥后, 将 Formvar 膜从网格周围撕裂, 并取下带有剖面的网格。</li>
	<li>在染色管的凹槽中设置网格16 , 按正确的顺序 (图 13), 并将铀醋酸盐和柠檬酸铅24的着色切片。 注: 有一个沟槽0.6 毫米深沿长轴的管切割与刀片。该管用于防止在使用镊子时的辅助薄膜发生意外破损, 因为在染色和洗涤过程中, 不需要直接处理网格,16。</li>
</ol>9. 对串行部分的观察 (图 14)
<ol><li>按照正确的顺序 (图 14a) 将 multi-specimen 托架中的5网格设置为垂直于试样夹轴的网格狭缝的长轴。网格的手柄 (箭头) 不必被切割, 因为 "网格毒贩" 不会触及手柄。</li>
	<li>用 "网格毒贩" (图 14b) 固定网格, 并将标本夹插入透射电子显微镜。 注意: 使用所有单元格部分 (图 15) 的低放大图像通常是有用的, 以便在拍摄目标单元格之前, 在没有污染和良好的固定状态下找到有趣的微生物或细胞。</li>
	<li>在高倍率下拍摄所有单元格中目标单元格的图片。</li>
</ol>

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作者没有什么可透露的。

这里提出的方法不需要昂贵的设备。它只需要一个铝架 (图 3)、三狭缝网格 (图 6c)、节保持环路 (图 10a)、water-surface 提升环路 (图11a) 和染色管 (图 13)。目前的方法有许多特点。试样的大部分用于调整试样的表面和刀口, 使其彼此平行。在将一组串行剖面检索到狭缝网格时, 将序列切片分成组, 以避免使用一对头发线分隔节的困难。"节保持环" 用于避免混合节组的顺序。"水表面提升回路" 用于确保剖面位于水的顶端, 并且首先接触网格, 以便将它们放置在栅格上的所需位置。铝架上的 Formvar 支撑膜用于使网格上的剖面恢复更容易, 并避免支撑膜起皱。采用染色管, 避免用镊子意外折断支撑膜。
最近, 一个自动的磁带收集 ultramicrotome 被开发了25 , 以收集部分自动扫描电子显微镜 (SEM) 成像的电子不透明的塑料磁带。此方法可以可靠地切割数千个超薄部分26, 这是目前方法所无法实现的。此外, 串行块面 (SBF) 27和聚焦离子束 (纤维蛋白原)-SEM28用于收集动物细胞和组织的3D 信息。在这些方法中, 用金刚石刀或聚焦离子束进行切片是在 SEM 显微镜下集成的, 并以完全自动的方式进行, 无需特殊技术。虽然这些方法是有用的, 设备是如此昂贵, 没有许多机构购买这些机器。此外, 由于这些方法采用扫描电镜的照片部分, 显微的分辨率低于目前的方法, 采用透射电子显微镜 (TEM)。这些方法目前无法解决单个核糖体微粒、微管、丝和 DNA 纤维。
对于微生物的 structome 分析和未知微生物的超微结构研究, 有必要观察细胞壁、细胞膜、线粒体、细胞核、细胞核膜、内质网、叶绿体, 质, 高尔基体仪器, 核糖体, 和丝状元素在较高的分辨率。连续超薄切片的 TEM 观察是在三维度中进行这种分析的几种方法之一。由于超薄切片在观察后保持不变, 可以重复观察同一区域;这不是可能的在 SBF 或说谎-SEM, 因为被观察的区域在观察以后丢失。这一特性对于研究深海微生物是很重要的, 在这种情况下, 必须先在低放大率下搜索有趣的生物体, 然后在高放大倍数下以连续的目标生物体进行拍照。
总之, 目前的研究可以简单、廉价、可靠的方式进行连续超薄切片, 这对电子显微镜下高分辨率微生物的 3D ultrastructual 研究至关重要。

适当的连续超薄切片技术是必不可少的研究细胞和细胞成分三维在电子显微镜水平。研究了酵母细胞周期中纺锤极体的动态变化, 并揭示了其在细胞周期和重复时间上的形态学改变1,2,3, 4,5。在 2006年, 我们创造了一个新词 ' structome ' 通过结合 "结构" 和 "-", 并定义为 ' 定量和三维结构信息的整个细胞在电子显微镜水平 ' 6,7。
通过 structome 分析, 需要连续超薄切片技术, 发现酵母细胞的酿酒酵母和外 dermatitidis有大约20万核糖体7,8, 一个大肠杆菌细胞有2.6万核糖体9, a 结核分枝杆菌细胞有1700核糖体10 , Myojin 螺旋菌只有300核糖体11。这些信息不仅可以用来估计每个生物体的生长速率, 也有助于识别物种的9。
此外, structome 分析导致发现一个新的有机体;Parakaryon myojinensis在日本海岸的深海中发现, 其细胞结构介于原和真核之间的中间,121314,15。目前, 连续超薄切片技术被认为是非常困难的, 需要很长时间才能掌握。在这项研究中, 我们开发了一个可靠的方法, 任何人都可以进行连续超薄切片无困难。

<table><tbody><tr><td>Formvar making apparatus</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>652</td><td>W 180 x D 180 x H 300 mm</td></tr><tr><td>Glass slide</td><td>Matsunami Co. Ltd., Osaka</td><td>-</td><td>76 x 26 x 1.3 mm</td></tr><tr><td>Aluminum rack with 4-mm holes</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>658</td><td>W 30 x D 25 x H 3 mm, Refer to this paper</td></tr><tr><td>Stereomicroscope</td><td>Nikon Co. Ltd., Tokyo</td><td>-</td><td>SMZ 645</td></tr><tr><td>LED illumination for stereomicroscope</td><td>Nikon Co. Ltd., Tokyo</td><td>-</td><td>SM-LW 61 Ji</td></tr><tr><td>Trimming stage</td><td>Sunmag Co.Ltd., Tokyo</td><td>-</td><td>Tilting mechanism equipped, Refer to this paper</td></tr><tr><td>LED illumination for trimming stage</td><td>Sunmag Co.Ltd., Tokyo</td><td>-</td><td>Refer to this paper</td></tr><tr><td>Ultrasonic trimming blade</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>5240</td><td>EM-240, Refer to this paper</td></tr><tr><td>Diamond knife for trimming</td><td>Diatome Co. Ltd., Switzerland</td><td>-</td><td>45&amp;deg;</td></tr><tr><td>Diamond knife for ultrathin sectioning</td><td>Diatome Co. Ltd., Switzerland</td><td>-</td><td>45&amp;deg;</td></tr><tr><td>Ultramicrotome</td><td>Leica Microsystems, Vienna</td><td>-</td><td>Ultracut S</td></tr><tr><td>Mesa cut</td><td>Leica Microsystems, Vienna</td><td>-</td><td>Mirror</td></tr><tr><td>0.5% Neoprene W solution</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>605</td><td></td></tr><tr><td>Special 3-slit nickel grid</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>2458</td><td>Refer to this paper</td></tr><tr><td>Special 3-slit copper grid</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>2459</td><td>Refer to this paper</td></tr><tr><td>Section-holding loop</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>526</td><td>Refer to this paper</td></tr><tr><td>Water-surface-raising loop</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>527</td><td>Refer to this paper</td></tr><tr><td>Staining tube</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>463</td><td>Refer to this paper</td></tr><tr><td>Multi-specimen holder</td><td>JEOL Co. Ltd., Tokyo</td><td>-</td><td>EM-11170</td></tr><tr><td>JEM-1400</td><td>JEOL Co. Ltd., Tokyo</td><td>-</td><td>Transmission electron microscope</td></tr></tbody></table>

a method for obtaining serial ultrathin sections of microorganisms in transmission electron microscopy

在透射电镜高倍率下观察三维细胞和细胞成分, 需要制备试样的超薄切片。虽然准备连续超薄的部分被认为是非常困难的, 它是相当容易的, 如果正确的方法是使用。本文介绍了一种安全获取微生物连续超薄切片的 step-by 步骤。该方法的要点是: 1) 使用试样的大部分, 调整试样的表面和刀口, 使其相互平行;2) 在狭缝网格上检索一组连续剖面时, 要在组中切割连续节, 避免在分离时使用一对头发束的困难;3) 使用 "节-保持回路", 避免混淆节群的顺序;4) 使用 "水表面提升回路", 并确保剖面位于水的顶端, 并首先接触网格, 以便将它们放置在栅格上所需的位置;5) 使用铝架上的支撑膜, 使其更容易恢复网格上的剖面, 避免支撑膜起皱;6) 使用染色管, 避免用镊子意外折断支撑膜。这种新的方法可以毫不费力地获得连续的超薄切片。该方法可以对3D 高分辨率的微生物细胞结构进行分析, 使用自动胶带收集 ultramicrotome 法和连续块面或聚焦离子束扫描电子显微镜是无法实现的。

在该协议中, 三-狭缝网格用于拾取串行段。网格是由镍或铜制成的。串行部分放置在中间狭缝上。双方的狭缝是必要的, 以查看的部分时, 他们拿起网格。在用镊子 (图 11d) 拾取时, 为了使网格与串行剖面保持平行, 手柄弯曲 (图 6c, 右)。一个小手柄有利于防止网格主体的弯曲, 在拾取时会引起严重的问题, 并防止在染色过程中掉节。该网格优于传统的 2.0 mm x 1.0 mm 孔网格。也就是说, 剖面由两个 0.4 mm 的金属棒直接支撑 (窄于1.0 毫米), 在三狭缝网格中由 Formvar 支持膜支撑, 而剖面仅由传统孔网格中的支撑膜支撑。因此, 使用三狭缝网格进行高倍率照相时, 可以防止试样漂移。
图 15显示了带有串行部分的五网格的低放大率视图。由于每个截面都粘在一起, 所以在更高的放大倍数下, 在下一节中很容易找到相同的单元格。图 16和17是单元格部分的序列图像的示例。由于在3狭缝网格上安全地支持节, 在高放大倍数 (x 5万) 上的照片很容易在没有试样漂移的情况下拍摄。脱氧核糖核酸2毫微米的纤维在这些研究被拍摄了9,12。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56235/56235fig1.jpg"> 图 1.Formvar 支持薄膜制作设备.在该装置的上部柱上, 有一半的玻片被浸在 Formvar 溶液中, 通过用橡皮球通过 "a" 来压出空气溶液。通过打开三路塞并通过 "b" 释放空气以减少压力, 解决方案从列中排出。(转载自山口和安达, 201119具有权限)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56235/56235fig2.jpg"> 图 2.从水中的玻璃滑梯上释放胶片的方法.(a) 玻璃片上的四边缘用刀片刮掉。(b) Formvar 膜在水上漂浮, 低角度缓慢降低玻璃滑动。(转载自山口和安达, 201119具有权限)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56235/56235fig3.jpg"> 图 3.Formvar 支持胶片在铝架上.(a) Formvar 膜从水中舀出铝架。(b) 机架存放在干燥, 直至使用。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56235/56235fig4v2.jpg"> 图 4.在显微镜下, 用超声波修剪刀片和刀片修剪试样块.(a) 在光学显微镜下观察标本块, 证实标本的存在。图中所示的块包含了与从深海收集的 chaeta (长度约1.7 毫米) 相关的微生物12,14。(b) 修剪阶段。(c) 显微镜的整体形象。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig4v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56235/56235fig5.jpg"> 图 5.用金刚石刀修整试样块切片.(a) 切割左边缘的图示。(b) 块面从试样的左边缘处切割约 100 nm 的位置。(c) 放置在刀台上的镜子 (台面切割, M)。(d) 试样表面。注意, 细长部分的上侧和下侧是平滑和完全平行的。另外请注意, 肩部被切割以标记切割方向 (参见图 8)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56235/56235fig6.jpg"> 图 6.在试样表面进行氯丁橡胶处理.(a) 为了把标本卡盘放回原来的位置, 切片的恰克和卡盘持有者用胶带标记, 并在边界处切割。(b) 用巴斯德吸管将试样润湿, 使用氯丁橡胶溶液。(c) 三-狭缝网格, 用于拾取串行段。手柄弯曲到60度 (c, 右)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56235/56235fig7.jpg"> 图 7.切片和木扶手的塑料盖。塑料盖有助于防止超薄切片中的气流。木扶手是用来做微妙的工作, 如检索串行部分。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56235/56235fig8v2.jpg"> 图 8.设置截面厚度和获得适当厚度的截面。在连续段数达到20后, 截面厚度设置为 10 nm。由于切片不能切割10纳米厚的部分, 没有新的节出现, 和以前的切割部分成为分离的刀口。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig8v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56235/56235fig9.jpg"> 图 9.通过协议获得的超薄剖面的示例.请注意, 切割后的五组长度约为 1.8 mm 的连续段已经分离。在这种特殊情况下, 超薄部分的中心的颜色是黄色的由于存在的标本。部分的其他部分, 不包含样品和仅由树脂组成, 是银色的在颜色。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56235/56235fig10.jpg"> 图 10."节-保持回路"。(a) 顶部视图 (上半部分) 和底部视图 (照片的下部)。(b) 节保持回路用于保存第三组连续段。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig10large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/56235/56235fig11.jpg"> 图 11.串行部分的检索。(a) "水表面抬高回路" (WSRL).该循环的手柄可以从轴上移除, 这是可移动的。通过转动螺钉顶部, 也可以上下移动循环。(b) WSRL 被磁铁固定在刀台上。(c) 更密切地看待 (b)。(d) 在检索过程中循环、水面、剖面和网格的图示 (侧面视图)。超薄剖面可以通过降低裸网格与超薄截面平行而精确地检索到狭缝网格。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig11large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/56235/56235fig12.jpg"> 图 12.将网格放置在 Formvar 胶片上.在铝架上的 Formvar 膜上, 放置截面的网格与一小滴水放在一起。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig12large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/56235/56235fig13.jpg"> 图 13.染色管16。一个凹槽0.6 毫米深是沿长轴使用刀片。网格按正确的顺序放置在凹槽中。管子的一端被切割 (箭头头) 以标记其方向。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig13large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/56235/56235fig14.jpg"> 图 14.将网格放置在标本架上。(a) 网格按正确的顺序放置在标本架上。(b) 然后用 "网格毒贩" 固定网格。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig14large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/56235/56235fig15.jpg"> 图 15.安装在狭缝网格上的串行部分。该图显示了安装在一个网格上的18到25节。每个狭缝中的数字表示从第一节开始的序列号。第一个节比其他部分更粗 (请参见图 8)。请注意, 这些截面连接在一起并具有相同的粗细。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig15large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 16" class="xfigimg" src="/files/ftp_upload/56235/56235fig16.jpg"> 图 16.Parakaryon myojinensis12 的串行部分.左下方的数字表示相对于第一节的序列。该图显示了 12 67 完整的部分。这个细胞被发现有一个大的核组成的裸 DNA 纤维, 一个单一的核膜, 和共生类似细菌, 但没有线粒体。因此, 这个有机体似乎是一个中间生命形式演变从原到核12。N, 核。转载自山口和女人, 2014。具有权限的14 。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig16large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 17" class="xfigimg" src="/files/ftp_upload/56235/56235fig17.jpg"> 图 17.大肠埃希菌9 的串行部分.左下方的数字表示相对于第一节的序列。该图显示了12完整的部分。发现该细胞含有21700核糖体9。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig17large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy at JoVE.com

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

本研究为在透射电子显微镜下获取无昂贵设备的微生物的连续超薄切片提供了可靠、简便的程序。

一种在透射电镜中获取微生物连续超薄切片的方法

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin ...

a-method-for-obtaining-serial-ultrathin-sections-microorganisms

Nanyang Technological University

Research

JoVE Journal

Engineering

14.1K 次观看.  Chiba University. 本研究为在透射电子显微镜下获取无昂贵设备的微生物的连续超薄切片提供了可靠、简便的程序。

视频: A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

线粒体和内质视网膜成像由相关光和体积电子显微镜

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

科研

生物化学

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

提高2D和3D皮肤<em&gt;体外</em&gt;模型使用大分子拥挤

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

生物工程

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

非人类灵长类动物脑组织的制备预嵌入免疫组化和电子显微镜

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

神经科学

An Unbiased Approach of Sampling TEM Sections in Neuroscience

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative studies of the changes in densities and distributions of such features, an unbiased sampling protocol is vital for reliable results. Here, we present a workflow for the image acquisition of brain samples. The workflow allows systematic uniform random sampling within a defined brain region, and the images can be analyzed using a disector. This technique is much faster than extensive examination of serial sections but still presents a feasible approach to estimate the densities and distributions of ultrastructure features. Before embedding, stained vibratome sections were used as a reference to identify the brain region under investigation, which helped speed up the overall specimen preparation process. This approach was used for comparative studies investigating the effect of an enriched-housing environment on several ultrastructural parameters in the mouse brain. Based on the successful use of the workflow, we adapted it for the purpose of elemental analysis of brain samples. We optimized the protocol in terms of the time of user-interaction. Automating all the time-consuming steps by compiling a script for the open source software SerialEM helps the user to focus on the main work of acquiring the elemental maps. As in the original workflow, we paid attention to the unbiased sampling approach to guarantee reliable results.

We introduce a novel workflow for electron microscopy investigations of brain tissue. The method allows the user to examine neuronal features in an unbiased fashion. For elemental analysis, we also present a script that automatizes most of the workflow for randomized sampling.

神经科学中透射电镜切片采样的一种无偏方法

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

对于样品制备与TEM分析低细胞数量的另一种方法

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

生物学

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

准备厚生物样品的观察扫描透射电子断层扫描

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique cristae-rich double membrane ultrastructure of this organelle is elegantly arranged to carry out multiple functions by partitioning biomolecules. Mitochondrial ultrastructure is intimately linked with various functions; however, the fine details of these structure-function relationships are only beginning to be described. Here, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle. Serial-section electron tomography may be adapted to study any cellular structure in three-dimensions.

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

In this protocol, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle.

连续切片电子断层扫描显示的 3D果蝇间接飞行肌的线粒体超微结构研究

Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique ...

Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ultrastructure of cells; artifacts and extraction of cell contents are usually observed. Rapid freezing is a better alternative for the preservation of cell structure. Sandwich freezing of living yeast or bacteria followed by freeze-substitution has been used for observing the exquisite natural ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells or human tissues has also been used to reveal the ultrastructure of cells and tissues. 
These studies have thus far been carried out with a handmade sandwich freezing device, and applications to studies in other laboratories have been limited. A new sandwich freezing device has recently been fabricated and is now commercially available. The present paper shows how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. Also shown is the preparation of specimens for ultrathin sectioning after rapid freezing and procedures for freeze-substitution, resin embedding, trimming of blocks, cutting of ultrathin sections, recovering of sections, staining, and covering of grids with support films.

Here we show how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. We also show how to prepare specimens for ultrathin sectioning after rapid freezing.

使用夹层冷冻装置快速冷冻，在电子显微镜下对生物标本进行良好的超微结构保存

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ...

Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy

Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is often limited to two dimensions, hampering the ability to fully describe the three-dimensional (3D) ultrastructure and functional relationship between organelles. Volume electron microscopy (vEM) describes a collection of techniques that enable the interrogation of cellular ultrastructure in 3D at mesoscale, microscale, and nanoscale resolutions. 
This protocol provides an accessible and robust method to acquire vEM data using serial section transmission EM (TEM) and covers the technical aspects of sample processing through to digital 3D reconstruction in a single, straightforward workflow. To demonstrate the usefulness of this technique, the 3D ultrastructural relationship between the endoplasmic reticulum and mitochondria and their contact sites in liver hepatocytes is presented. Interorganelle contacts serve vital roles in the transfer of ions, lipids, nutrients, and other small molecules between organelles. However, despite their initial discovery in hepatocytes, there is still much to learn about their physical features, dynamics, and functions. 
Interorganelle contacts can display a range of morphologies, varying in the proximity of the two organelles to one another (typically ~10-30 nm) and the extent of the contact site (from punctate contacts to larger 3D cisternal-like contacts). The examination of close contacts requires high-resolution imaging, and serial section TEM is well suited to visualize the 3D ultrastructural of interorganelle contacts during hepatocyte differentiation, as well as alterations in hepatocyte architecture associated with metabolic diseases.

A simple and comprehensive protocol to acquire three-dimensional details of membrane contact sites between organelles in hepatocytes from the liver or cells in other tissues.

使用连续截面电子显微镜对肝细胞中组织间接触位点进行三维表征

Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

用于解释霉菌异养植物组织中真菌定植和种子共生萌发的显微镜技术

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...